Andreas Lamanda (PhD), Vinzenz Bieri (PhD) and Melek DilekTurgut (Dr. med. dent.)
The aim of the present study was to investigate whether amyloglucosidase in combination with lysozyme, hydrogencarbonate and di-hydrogenphosphate could be able to provide similar buffer attributes as the human saliva. Therefore the buffer attributes (power, range, optimum and value) of a model system for salivary protein buffering, containing the two proteins, amyloglucosidase and lysozyme, in combination with hydrogencarbonate, di-hydrogenphosphate were quantified The protein containing model system was found to have almost the same buffer attributes as saliva. As in saliva neither the buffer optimum nor the buffer value at pH 4.3 could be derived from hydrogencarbonate and/or di-hydrogenphosphate, it was concluded that buffering found in human saliva at pH 4.3 could be ascribed to the salivary proteins.
When food enters our alimentary canal through the mouth, it first comes into contact with saliva, mainly excreted by the three major salivary glands. The salivary buffer capacity is a natural physiological protection aiming at keeping a supersaturated state with respect to enamel and dentine near the tooth surface; both in relation to caries (1) and dental erosion (2,3). Enamel and dentine are composed primarily of a carbonate substituted calcium deficient hydroxyapatite. When hydroxyapatite is in contact with water (saliva), hydroxyl ions (OH–) can remove from the tooth surface during an erosive challenge like drinking an apple juice (4), vomiting (5) or gastro-oesophageal reflux (6). If this process is repeated frequently, a loss of tooth substance, also known as erosion, may be the consequence (7,8). Dissolution ends and remineralisation of the dental hard tissues occurs when the pH in close proximity to the tooth begins to rise (3). This rise in pH is caused by saliva that permanently covers the structures forming the oral cavity. The salivary components responsible for the increase in pH are the three buffer systems, carbonate, phosphate and protein buffer system(9,10). The carbonate and phosphate systems have been well characterized (9,11-14). With the exception of the knowledge regarding that the total protein concentration varies from 0.15 to 0.65% (15) and that 940 different protein species are present in saliva (16-23), the information about the protein buffer system is scarce (24-28).
Over the last 40 years, the prevalence of dental erosion increased continuously (29,30). As the buffer characteristics of saliva can influence the erosion process (4), the aim of the present study was to quantify the buffer characteristics of a model system for salivary protein buffering by incorporating di-hydrogenphosphate, hydrogencarbonate and the two proteins amyoglucosidase and lysozyme.
MATERIALS & METHODS
Five mM di-hydrogenphosphate: 0.68 g KH2PO4 (Merck, for analysis, Dietikon, Switzerland, 136.09 g/mol, pKa=7.18 at 35°C, (31)) was dissolved in 1000 ml deionised water. 10 mM hydrogencarbonate: 0.84 g NaHCO3 (Merck, for analysis, 84.01 g/mol, pKa=6.1 at 37°C, (12)) was dissolved in 1000 ml deionised water. Ten mM hydrogencarbonate and 5 mM di-hydrogenphosphate: 0.68 g KH2PO4 and0.84 g NaHCO3 were dissolved in 1000 ml deionised water. Ten mM (0.1%) Amyloglucosidase: amyloglucosidase from Aspergillus niger (Fluka, BioChemika, Buchs, Switzerland, Swissprot P69328, 640 amino acids, Mr=98 kDa, pI=4.35) was used as a model for human a-amylase, (Swissprot P04745, 511 amino acids, Mr=57.8 kDa, pI=6.4). Ten mg amyloglucosidase was dissolved in 10 ml deionised water. Three hundred and fourty mM (0.5%) lysozyme: lysozyme from hen egg white (Fluka, BioChemika, Swissprot P00698, 147 amino acids, Mr=14.6 kDa, pI=9.4), was used as a model for human salivary lysozyme (Swissprot P61626, 148 amino acids, Mr=16.5 kDa, pI=9.4). Fifty mg lysozyme was dissolved in 10 ml deionised water. Ten mM (0.1%) amyloglucosidase and 340 mM (0.5%) lysozyme: 10 mg amyloglucosidase and 50 mg lysozyme were dissolved in 10 ml deionised water. Ten mM (0.1%) amyloglucosidase, 340 mM (0.5%) lysozyme, 10 mM hydrogencarbonate and 5 mM di-hydrogenphosphate: 10 mg amyloglucosidase and 50 mg lysozyme were dissolved in 10 ml of a solution containing 0.68 g KH2PO4 and0.84 g NaHCO3 per 1000 ml deionised water. After adjustment of the pH to 7, all solutions were stored in gas-proof closed vessels.
Ten ml of the analytes (saliva sample or solutions) were placed in a vessel in a water bath and stirred at 37°C. First, 5 ml of NaOH 0.01 mol/l were added in steps of 200 ml to enclose the buffer range of di-hydrogenphosphate (pH 6.1-8.1) and then 25 ml of HCl 0.01 mol/l were added in steps of 200 ml. The pH was recorded after each addition step so that 150 pH measurements per titration were performed. The buffer power B [mol/l] was calculated according to the formula B=c2/2c, where c is the concentration in mol/l of the buffer component(s) (32). Data points were fitted with Sigmaplot V9.0 (Systat Software, Erkrath, Germany) . Buffer values b in [mol/l×pH=slyke (33)] were calculated as b=–DC/DpH (32) where DC is the amount of the titrator used (acid/base) and DpH is the change in pH caused by the addition of the titrator. To calculate the inflection coordinates of the titration curves, the concentration and pH were normalized. Inflections were read out of a plot of DpH/DC against the pH (first derivative). The buffer power B was quantified at the inflections (Ia to Id).
The buffer power, in mmol (H+) and mmol (OH-) was used to quantify the amount of acid and base. Calculated values were compared to those that were measured experimentally. The buffer range, in pH units, was used to describe the pH interval where the buffering reaction of a certain compound takes place. The buffer optimum, at a certain pH, was used to define the pH of the highest buffering within the buffer range. The buffer value, in slyke (=mol/l×pH), was used to quantify the buffer capacity.
Human saliva samples
The saliva was collected using a widely accepted procedure (34) under resting conditions, between 9:00 am and 10:00 am, from unmedicated volunteers who refrained from eating, drinking, smoking and performing oral hygiene measures for 2 h before collection. Prior to saliva collection, the procedures were explained to the patients and an informed consent was taken from each of them. After collection, the buffer capacity of the saliva samples was determined using the CRTbuffer test (Ivoclar Vivadent, Schaan, Lichtenstein) according to the manufacturer’s protocol. The samples were subjected to titration immediately after collection to prevent discrepancies caused by protease activity and the formation of ammonium by urease.
Averaging saliva titration curves
Unstimulated saliva samples of 5 male subjects aged 35 to 45 with buffer capacities ranging from low to high according the CRTbuffer test, were subjected to acid base titration as described above. The pH measurements were recorded with equal spaces and constant increments due to the monotone nature of the titration. As all pH measurement points in the saliva titration curves corresponded to each other, they were averaged and the standard deviation was calculated.
Search for model proteins
First, the availability (>1g) of high-purity water soluble proteins at reasonable costs (<100 USD/g), was checked. In this regard, about 100 proteins were selected. Then, the isoelectric point (theoretical best buffering point) of the selected proteins was calculated with the ProtParam analysis tool (35) at www.expasy.org. The proteins with an isoelectric point in a pH range from pH 3 to 5 or pH 8 to 10 were selected, which contained only proteins with buffer optima beyond the buffer range of hydrogencarbonate and di-hydrogenphosphate (group A).
Secondly, a list, containing all known human salivary proteins was created from the literature. Theirisoelectric points were calculated with the ProtParam analysis tool (35). The proteins with an isoelectric point in a pH range from pH 3 to 5 or pH 8 to 10 were selected, containing only proteins with buffer optima beyond the buffer range of hydrogencarbonate and di-hydrogenphosphate (group B). The amino acid sequences of the proteins in group A were aligned against the amino acid sequences of the proteins in group B with BLAST (36) or LALIGN (37) at www.expasy.org/tools/. A sequence in group A was selected if more than 30% of its sequence was identical to the sequence in group B.
Inorganic buffer characteristics
A solution of 5 mM di-hydrogenphosphate (Fig. 1,a) was found to have buffer power of 30 mmol acid (hydrogen ions, H+) and 24 mmol base (hydroxyl ions, OH-). Optimal buffering was measured at pH 6.7 with 0.003 slyke. The calculated buffer power was 25 mmol acid and base. A solution of 10 mMhydrogencarbonate(Fig. 1,b) was found to have a buffer power of 74 mmol (H+) and 8 mmol (OH-). Optimal buffering was measured at pH 6.2 with 0.005 slyke. The calculated buffer power was 50 mmol acid and base.A solution of 10 mM hydrogencarbonate plus 5 mM di-hydrogenphosphate (Fig. 1,c), was found to have a buffer power of 110 mmol (H+), and 22 mmol (OH-). The distances between inflections Ia and Ib as well as between Ib and Ic were larger than the calculated 25 mmol (H+). Optimal buffering was measured at pH 6.5 with 0.008 slyke. The calculated buffer power was 75 mmol acid and base.
Organic buffer characteristics
Search for salivary a-amylase and lysozyme substitutes
Fifteen proteins fitted to the selection criteria for group A and 346 proteins for group B. Based on the pI selection criteria and the sequence alignments, two proteins were chosen to serve as model proteins: Lysozyme from hen egg which has an isoelectric point of 9.4 and 57% sequence similarity to human salivary lysozyme and amyloglucosidase from Aspergillus niger which has an isoelectric point of 4.35 and 35% sequence similarity to human a-amylase.
A solution of 10 mM (0.1%) amyloglucosidase in water (Fig. 2, f) was found to have a buffer power of 68 mmol (H+) and 0 mmol (OH-). The buffer range spanned from pH 3.3 to 5.3 with optimal buffering at pH 4.35 and a buffer value of 0.004 slyke. A solution of 340 mM (0.5%) lysozyme in water (Fig. 2,e) was found to have no measurable buffer attributes, although the protein has 32 titrable groups (38). A solution of 10 mM (0.1%) amyloglucosidase and 340 mM (0.5%) lysozyme in water (Fig. 2,g) was found to have a buffer power of 84 mmol (H+) and 0 mmol (OH-). The buffer range spanned from pH 3.3 to 5.3 with an optimal buffering at pH 4.5 and a buffer value of 0.007 slyke.
Combined inorganic and organic buffering characteristics
A solution of 10 mM (0.1%) amyloglucosidase and 340 mM (0.5%) lysozyme, 10 mM hydrogencarbonate and 5 mM di-hydrogenphosphate (Fig. 1, 2, 3A, 3B curve d) was found to have a buffer power of 158 mmol (H+) and 38 mmol of base. There were two discrete buffer optima within the buffer range between pH 3.4 to 7.5. The first was at pH 4.3 with a buffer value of 0.005 slyke, whereas the second was at pH 6.5 with a buffer value of 0.01 slyke. Human unstimulated whole saliva (Fig. 3A,h) was found to have a buffer power of 168 mmol (H+) and 42 mmol (OH-). Human unstimulated whole saliva had a buffer zone from pH 3.4 to 8 with buffer values starting from 0.005 slyke at pH 3.4 ascending to 0.01 slyke at pH 6.5 and remaining constant at 0.01 slyke until pH 8. The average of the human unstimulated whole saliva collected from 5 individuals (Fig. 3B,j) was found to have a buffer power of 154 mmol (H+) and 36 mmol (OH-). Average human unstimulated whole saliva had a buffer zone from 3.5 to 8 with buffer values starting with 0.004 slyke at pH 4 ascending to 0.008 slyke at pH 6.5 and descending to 0.003 slyke until pH 8.
In the present study, the buffer attributes (power, range, optimum and value) of a model system for salivary protein buffering were quantified. In the first step, the procedure was done by dissolving each of the model compounds in water separately. In the second step, the same compounds having the concentration as in the human saliva were mixed. In the third step, the procedure was done with human saliva and the measured data were compared to the model system. Amyloglucosidase from Aspergillus niger and lysozyme from hen egg were used as model proteins because purified genuine or recombinant expressed salivary proteins were not available in the desired purity and quantity. Amyloglucosidase and lysozyme have the ideal physicochemical properties to demonstrate buffering beyond the buffer ranges of di-hydrogenphosphate and hydrogencarbonate. Both proteins have a high amino acid sequence similarity to their human counterparts whereas lysozyme from hen egg has almost the same molecular mass and the same isoelectric point as human salivary lysozyme. Moreover, this approach was feasible as the buffer function of a protein is dependent on its isoelectric point, but independent of its catalytic properties or the species where it originates from.The total amyloglucosidase and lysozyme concentration (0.6%) used in the experiments, did not exceed the total protein concentration found in human saliva (15). Three hundred and forty six human salivary proteins had their buffer optima beyond the buffer range of hydrogencarbonate and di-hydrogenphosphate (pH 5.1 to 8.1) what pointed out the plausibility that buffering beyond pH 5.1 to 8.1 could be based on proteins. Finally, buffering from proteins in saliva is likely to occur as in the rest of the human body proteins are the most potent buffer substances (39).
In the present study, the experimentally determined buffer attributes of 5 mM di-hydrogenphosphate and 10 mM hydrogencarbonate were in agreement with the published data (9,32). The observation that the carbonate system buffered 48% more acid than expected by calculation. was attributed to the open system that was used for the titration experiments and is in agreement with published data (33,40).
Human whole saliva had a buffer zone spanning from pH 3.4 to 8 compassing the buffer ranges of hydrogencarbonate (pH 5.1 to 7.1) and di-hydrogenphosphate (pH 6.2 to 8.2). However, buffering in the range of pH 3.4 to 5 was not attributed to the buffering of hydrogencarbonate or di-hydrogenphosphate. It is known that at pH 4.3, hydrogencarbonate and di-hydrogenphosphate exhibit a maximum of 3% of their optimal buffer values (32).
The results of this study showed that the buffer value of the model system was 20 times higher than expected from 5 mM di-hydrogenphosphate and 10 mM hydrogencarbonate. However, at pH 4.3, the model system had exactly the same buffer value as human saliva and a buffer power that varied very little compared to the human saliva. The total protein concentration used in this study was at the upper limit of the salivary protein concentration found in the literature. However amyloglucosidase was used in lower concentration (0.1%) than lysozyme (0.5%). Eighty one percent of the protein buffer power at pH 4.3 was based in the model system on amyloglucosidase. Therefore the majority of the protein buffer power was based on amylogluycosidase whose concentration did not exceed the protein content typically found in resting saliva (9,11). As the buffer values of the saliva measured in this study were in agreement with those published by Bardow et al. (9) and even high concentrations of di-hydrogenphosphate and/or hydrogencarbonate can exhibit little buffer effect at pH 4.3 (32), it would be reasonable to conclude that we had the evidence that salivary buffering at pH 4.3 could be derived from the proteins.
For the combination of hydrogencarbonate and di-hydrogenphosphate with amyloglucosidase and lysozyme, 75% of the buffer value at pH 6.5 derived from hydrogencarbonate and di-hydrogenphosphate. The remaining 25% derived from amyloglucosidase and lysozyme. These results were unexpected as the fraction of the buffer value derived from proteins were responsible for only 3% of the buffer value at pH 6.5 (32). Therefore, these findings were concluded as the evidence of the contribution of proteins to a larger fraction of the buffer value at pH 6.5 than hitherto assumed. These results, therefore, both support the hypothesis of Sellmann (41) regarding that proteins buffer at low pH values and the assumption of Freidin et al.(26) who proposed protein buffer activity in a zone from pH 5.5 to 7.8. There might have been two reasons why Lilienthal could not obtain a buffer capacity from dialyzed saliva. On one hand, a titration method with relatively low sensitivity was used (24). On the other hand, the protein preparation method applied, facilitated basic protein hydrolysis and therefore destruction of the proteins (42).
In this study, the role of carbonic anhydrases and urease was neglected because both enzymes are found mainly in the enamel pellicle (10,43) which was not included in the experiments.The present study of a model system for protein buffering demonstrated that salivary buffering between pH 3.4 and 5 was not based on hydrogencarbonate and di-hydrogenphosphate but rather on proteins. The high analogy between the pH 4.3 buffer values of whole saliva and the model system is a clear evidence for protein buffering. However, the discrete buffer optimum found in the model system cannot explain the existence of the detected buffer zone (pH 3.4 to 8) in saliva. Buffering between pH 5.1 and 8 was found to be based mostly on hydrogencarbonate and di-hydrogenphosphate but also seemed to be dependent on a larger fraction of proteins than thought before (9,24). In this context it is worth mentioning the recently discovered human salivary a-amylase subproteom which consists of 67 amylase subspecies with isoelectric points ranging from pH 3.5 to 7.6 (44). These a-amylase variants may provide, like zwitterionic buffers (45,46), a buffer system operational between pH 3.5 and 5 and auxiliary buffering through anionic and cationic sites present as non-interacting carboxylate and ammonium side chains between pH 5 and 8.
We thank Dr. Vera Navdaeva from the Department of Chemistry and Biochemistry, University of Bern, for the translation of the article from Freidin et al. (26) that was originally written in Russian and Dr. Vinzenz Bieri for the help with the manuscript. Parts of this article overlap with the article Protein Buffering in Model Systems and in Whole Human Saliva of Lamanda A, Cheaib Z, Turgut MD published in PLoS ONE 2(2): e263. doi:10.1371/journal.pone.0000263. The article published in PLoS One is based on this article where we laid the fundament about protein buffering in human saliva. In the present article we had to speculate about the protein buffer system. Only a short time later we found evidence for these speculations.
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